ONCOLOGY LETTERS 12: 4949-4956, 2016 The biological significance of methylome differences in human papilloma virus associated head and neck cancer MARIA J. WORSHAM1, KANG MEI CHEN1, INDRANI DATTA2, JOSENA K. STEPHEN1, DHANANJAY CHITALE3, ALEXANDRA GOTHARD4 and GEORGE DIVINE2 Departments of 1Otolaryngology/Head and Neck Research, 2Public Health Sciences and 3Pathology, Henry Ford Hospital, Detroit, MI 48202; 4Department of Biology, Kalamazoo College, Kalamazoo, MI 49006, USA Received May 31, 2016; Accepted August 26, 2016 DOI: 10.3892/ol.2016.5303 Abstract. In recent years, studies have suggested that Introduction promoter methylation in human papilloma virus (HPV) posi- tive head and neck squamous cell carcinoma (HNSCC) has The results from a number of studies involving next‑generation a mechanistic role and has the potential to improve patient sequencing sequence analysis, gene expression microarrays, survival. The present study aimed to replicate key molecular whole‑exome sequencing and genome‑wide methylation findings from previous analyses of the methylomes of HPV studies support the observation that human papilloma virus positive and HPV negative HNSCC in an independent cohort, (HPV) positive squamous head and neck cancer (HNSCC) to assess the reliability of differentially methylated markers in is a distinct entity and therefore exhibits a particular set of HPV-associated tumors. HPV was measured using real-time somatic alterations (1-6). Data from epidemiological and quantitative PCR and the biological significance of methyla- clinical studies have also indicated that HPV positive HNSCC tion differences was assessed by Ingenuity Pathway Analysis is clinically different from HPV negative HNSCC in terms of (IPA). Using an identical experimental design of a 450K patient characteristics, sensitivity to treatment and molecular methylation platform, 7 of the 11 genes were detected to be biology (7,8). significantly differentially methylated and all 11 genes were Global characterization of the HNSCC methylome either hypo- or hypermethylated, which was in agreement demonstrates the different landscapes in HPV positive tumors with the results of a previous study. IPA's enriched networks compared with HPV negative tumors. CpG methylation is analysis identified one network with msh homeobox 2 (MSX2) higher in HPV positive cells in both repetitive and non-repet- as a central node. Locally dense interactions between genes in itive regions (genic and non-genic) (9,10). Methylome analysis networks tend to reflect significant biology; therefore MSX2 of HPV positive and HPV negative primary formalin‑fixed was selected as an important gene. Sequestration in the top paraffin‑embedded tumors using the Illumina HumanMeth- four canonical pathways was noted for 5‑hydroxytryptamine ylation450K Beadchip platform (2) have confirmed previous receptor 1E (serotonin signaling), collapsin response mediator reports (9,11) that the DNA methylation signature of HPV protein 1 (semaphorin signaling) and paired like homeodo- positive HNSCC is different from that of HPV negative main 2 (bone morphogenic protein and transforming growth HNSCC and that HPV positive HNSCC has a strong tendency factor‑β signaling). Placement of 9 of the 11 genes in highly to undergo hypermethylation. A previous study (2) highlighted ranked pathways and bionetworks identified key biological 11 genes: Chromosome 14 open reading frame 162/coiled-coil processes to further emphasize differences between HNSCC domain containing 177 (C14orf162/CCDC177), cadherin 8 HPV positive and negative pathogenesis. (CDH8), collapsin response mediator protein 1 (CRMP1), engulfment and cell motility 1 (ELMO1), 5‑hydroxytrypta- mine receptor 1E (HTR1E), meiotic double-stranded break formation protein 1 (MEI1), msh homeobox 2 (MSX2), protocadherin 10 (PCDH10), protocadherin β-11 (PCDHB11), paired like homeodomain 2 (PITX2) and synapsin II (SYN2) as undergoing significantly different methylation processes in HPV positive and HPV negative HNSCC (2) (Table I). Correspondence to: Dr Maria J. Worsham, Department of It has been established that patients with HPV posi- Otolaryngology/Head and Neck Research, Henry Ford Hospital, 1 Ford Place, 1D Detroit, MI 48202, USA tive HNSCC have better survival outcomes than those with E‑mail: [email protected] HNSCC that are HPV negative, possibly due to improved responses to chemoradiation (12). However, it remains unclear Key words: differential methylation, pathway analysis, canonical why HPV positive patients experience improved prognoses‑the pathways, networks underlying mechanisms responsible for these outcomes have not yet been investigated. A mechanistic role for promoter methylation is beginning to be established, with the aim of 4950 WORSHAM et al: BIOLOGICAL SIGNIFICANCE OF METHYLOME DIFFERENCES IN HPV‑ASSOCIATED H&N CANCER improving survival outcomes of patients with HPV positive normalized and raw data were submitted to the GEO (Gene HNSCC (13). The aim of the current study was to verify that Expression Omnibus, NCBI) according to the instructions the 11 aforementioned genes undergo significantly different provided (GEO accession numbers: GSE67114). The resulting methylation in HPV positive and HPV negative HNSCC β‑values were exported to Microsoft Excel, JMP and SAS tumor samples and to assess their biological significance using (SAS Institute, Inc., Cary, NC, USA) for data analysis. Ingenuity Pathway Analysis (IPA) for biomarker potential in HPV-associated HNSCC. Pathway analysis. To determine the biological processes occurring within the 11 differentially methylated genes, Materials and methods they were uploaded to the Ingenuity pathway Analysis (IPA; Ingenuity Systems, Inc; Qiagen, Inc.). IPA integrates genes DNA extraction and amplification. DNA from 4 HPV posi- and molecules that are part of the same biological functions tive and 4 HPV negative freshly frozen (‑80˚C) primary or regulatory networks interacting together. HNSCC tumor samples, resected from patients admitted between December 2010 and January 2012 in the Depart- Statistical methods. There was a priori evidence that the ment of Otolaryngology/Head and Neck Surgery (Henry Ford 11 genes were differentially methylated and each in a partic- Hospital, Detroit, USA), were subjected to comprehensive ular direction, therefore, P<0.05 was considered to indicate a genome‑wide methylation profiling using the Infinium Human- statistically significant difference. Methylation450K BeadChip kit (Illumina, Inc., San Diego, CA, USA). Tumor site and demographic characteristics are Results presented in Table II. For the present study, patient informed consent for inclusion of surgically resected tissues was given, HumanMethylation450K BeadChip analysis. For all genes, and the study was approved by the Henry Ford Health System 99.97% of the CpGs and 100% of the CpGs associated with Institutional Review Board committee. the 11 genes of particular interest, met the detection threshold DNA was extracted according to the manufacturer's of P<0.05 and therefore were eligible for analysis. protocol (Qiagen, Inc., Valencia, CA, USA). Tumor HPV Of the 11 genes of interest, the methylation status of 7 DNA concentrations were measured using real‑time quanti- differed significantly between HPV positive and HPV nega- tative PCR (qPCR) as previously described (14,15). Briefly, tive HNSCC (Table I). CDH8, PCDHB11, ELMO1, MSX2, primers and probes to a housekeeping gene (β-globin) were and HTR1E were significantly hypermethylated and MEI1 run in parallel to standardize the input DNA. By using serial and C14orf162/CCDC177 were significantly hypomethyl- dilutions, standard curves were developed for the HPV viral ated (Fig. 1). The methylation status of all 11 genes as either copy number using CaSki (American Type Culture Collection, hypo‑ or hypermethylated was consistent with the results of a Manassas, VA, USA) cell line genomic DNA, which have previous study by Lechner et al (2) even though the methyla- 600 copies/genome equivalent (6.6 pg of DNA/genome). The tion of four genes did not differ significantly (P>0.05). cut‑off value for HPV16 positive status was >0.03 (>3 HPV genome copy/100 cells) (14-16). Pathway analysis. C14orf162/CCDC177 was excluded from The Infinium HumanMethylation450 BeadChip kit the list of genes in IPA as it was unaccounted for in the IPA includes 485,764 cytosine positions in the human genome knowledge database. IPA connected 7/10 genes in a 35 gene and covers 99% of Reference Sequence genes. The Reference network characterized by the following functions: Cellular Sequence collection aims to provide an integrated, comprehen- Development, Skeletal and Muscular System Development sive, well‑annotated non‑redundant set of sequences, including and Function, and Embryonic Development as the sole genomic DNA, transcripts and proteins (17). The interrogated enriched top network (Fig. 2). The seven genes exhibiting CpG sites are distributed among all 23 human chromosome significantly different methylation were MSX2, CRMP1, pairs (17). ELMO1, CDH8, SYN2, PCDH10 and PITX2 (Fig. 1). The top five molecular and cellular functions included Processing samples for the Infinium HumanMethylation450. cell morphology, cellular assembly, cellular movement, The Infinium HumanMethylation450 assays were performed cell-to-cell signaling and interaction and cell death and at the Applied Genomics Technology Center, Wayne State survival (Table III). ELMO1 was represented in all the top University (Detroit, MI, USA). Following